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The proinflammatory activity of recombinant serum amyloid A is not shared by the endogenous protein in the circulation.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 6, June 2010, pp 1660–1665
DOI 10.1002/art.27440
© 2010, American College of Rheumatology
The Proinflammatory Activity of
Recombinant Serum Amyloid A Is Not Shared by
the Endogenous Protein in the Circulation
Lena Björkman,1 John G. Raynes,2 Chandrabala Shah,2 Anna Karlsson,1
Claes Dahlgren,1 and Johan Bylund1
L-selectin was not down-regulated on RA patient neutrophils as compared with neutrophils from healthy
controls. Spiking SAA-rich whole blood samples from
RA patients with rSAA, however, resulted in L-selectin
shedding. In addition, SAA purified from human
plasma was completely devoid of neutrophil- or
macrophage-activating capacity.
Conclusion. The present findings show that rSAA
is proinflammatory but that this activity is not shared
by endogenous SAA, either when present in the circulation of RA patients or when purified from plasma
during an acute-phase response.
Objective. Elevated serum levels of the acutephase protein serum amyloid A (SAA) are a marker for
active rheumatoid arthritis (RA), and SAA can also be
found in the tissues of patients with active RA. Based on
a number of studies with recombinant SAA (rSAA), the
protein has been suggested to be a potent proinflammatory mediator that activates human neutrophils, but
whether endogenous SAA shares these proinflammatory
activities has not been directly addressed. The present
study was undertaken to investigate whether SAA in the
plasma of patients with RA possesses proinflammatory
properties and activates neutrophils in a manner similar to that of the recombinant protein.
Methods. Neutrophil activation was monitored by
flow cytometry, based on L-selectin shedding from cell
surfaces. Whole blood samples from healthy subjects
and from RA patients with highly elevated SAA levels
were studied before and after stimulation with rSAA as
well as purified endogenous SAA.
Results. Recombinant SAA potently induced
cleavage of L-selectin from neutrophils and in whole
blood samples. Despite highly elevated SAA levels,
The acute-phase protein serum amyloid A (SAA)
is present in the bloodstream at concentrations below
1 ␮M (corresponding to ⬍11 mg/liter) under physiologic
conditions, but levels increase significantly during the
acute-phase response following infection or conditions
of inflammation (1). For example, SAA levels detected
in peripheral blood of patients with rheumatoid arthritis
(RA) may be well above 50 ␮M (2). In accordance with
this, an elevated SAA level in serum is often used as a
marker of rheumatic diseases (3). A consequence of
long-term elevated levels of SAA is the risk of developing amyloid deposits in the tissues, leading to progressive
organ failure (4). In humans, SAA is a family of small
apolipoproteins of ⬃12 kd encoded by 4 different genes,
of which only 3 are known to be expressed. SAA1 and
SAA2 are major acute-phase proteins that are coordinately induced within a few hours during an acute-phase
response, and the mature protein sequences share
⬎90% amino acid identity (5).
A recombinant SAA1 (rSAA1) has been reported to have cytokine-like properties (6), but most of
the literature describing proinflammatory activities has
been generated from studies using a commercially avail-
Supported by the Swedish Medical Research Council, King
Gustaf V’s Memorial Foundation, the Gothenburg Medical Society,
the Swedish Society for Medicine, the Inga Britt and Arne Lundberg
Research Foundation, and the Swedish State under the LUA/ALF
agreement. Dr. Shah’s work was supported by an MRC PhD studentship.
1
Lena Björkman, MD, Anna Karlsson, PhD, Claes Dahlgren,
PhD, Johan Bylund, PhD: Sahlgrenska Academy, University of Gothenburg, Gothenburg Sweden; 2John G. Raynes, PhD, Chandrabala
Shah, PhD: London School of Hygiene and Tropical Medicine,
London, UK.
Address correspondence and reprint requests to Lena Björkman, MD, Sahlgrenska Academy, University of Gothenburg, Department of Rheumatology and Inflammation Research, Guldhedsgatan
10A, 413 46 Gothenburg, Sweden. E-mail: lena.i.bjorkman@
vgregion.se.
Submitted for publication September 16, 2009; accepted in
revised form February 23, 2010.
1660
SERUM AMYLOID A LACKS PROINFLAMMATORY ACTIVITY
able recombinant molecule constructed as a mixture of
human SAA1 and SAA2. We have, for instance, shown
that rSAA activates human neutrophils, and even
though the responsible receptor is currently unknown
(7), the concentrations of rSAA required for cell activation in vitro (1–10 ␮M) are well below the in vivo levels
found in the circulation of RA patients. Since neutrophils are clearly activated by rSAA at low micromolar
concentrations, we reasoned that circulating neutrophils
from RA patients should display an activated phenotype. We used a sensitive assay that enabled us to
monitor neutrophil activation in whole blood and found
no evidence of activation, despite high levels of endogenous SAA in the circulation. In contrast, addition of
rSAA to blood potently activated neutrophils, regardless
of background levels of SAA. In accordance with this, we
found that SAA purified from serum during an acutephase response was completely devoid of neutrophilactivating capacity and also lacked any proinflammatory
effect when added to adherent peripheral blood mononuclear cells (PBMCs). Our data demonstrate that the
SAA present in the circulation during an acute-phase
response is functionally different from the recombinant
molecule.
1661
pital, using an ELISA kit from BioSource. In parallel, blood
samples were collected from healthy volunteers. All blood
samples were drawn into heparinized tubes at room temperature and used immediately.
Determination of L-selectin expression. The exposure
of CD62L (L-selectin) on neutrophils was assessed by immunostaining and fluorescence-activated cell sorter analysis.
Briefly, complete heparinized blood (90 ␮l/tube) was incubated with or without stimulation for 20 minutes at 37°C, after
which 2 ml of ice-cold FACS Lysing Solution (Becton Dickinson) was added and samples stored on melting ice for 15
minutes. After washing, the cell pellet was incubated with
anti–L-selectin antibody (10 ␮l/cell pellet) and analyzed by
flow cytometry (FACScan; Becton Dickinson). At least 10,000
events were acquired, and neutrophils were gated on the basis
of side and forward scatter. All data were analyzed using
WinMDI 2.8 software.
TNF production from adherent PBMCs. Human
mononuclear cells were isolated by centrifugation over Histopaque 1077, and cells (3 ⫻ 106/ml) were adhered to plastic
for 2 hours in serum-free medium. Cells were incubated in
RPMI 1640 with 10% fetal calf serum for 6 days before
stimulation with control medium, lipopolysaccharide (1 ␮g/
ml), or SAA. Supernatants were collected at 24 hours, centrifuged, and assayed for TNF by ELISA.
Statistical analysis. One-way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test or
two-way ANOVA followed by Bonferroni correction was used
for statistical evaluation as appropriate. P values less than 0.05
were considered significant.
PATIENTS AND METHODS
Reagents. Human rSAA (PeproTech) was dissolved in
water and stored at ⫺70°C until use; endotoxin levels were
⬍0.1 ng/␮g (1 EU/␮g). Endotoxin levels were also tested by
standard methods at a clinical bacteriology laboratory (Sahlgrenska University Hospital, Gothenburg, Sweden), and preparations were found to contain ⬍0.056 EU/␮g. Tumor necrosis
factor (TNF) was from Sigma. Phycoerythrin-conjugated monoclonal antibody specific for CD62L (anti–L-selectin) was from
BD Biosiences, as was the TNF enzyme-linked immunosorbent
assay (ELISA) kit.
Endogenous SAA was purified as described previously
(8), starting with plasma obtained at plasmapheresis during an
acute phase response. Briefly, an SAA ELISA was used to
identify fractions containing SAA in sequential stages of
hydrophobic interaction chromatography on octyl-Sepharose,
gel filtration on Sepharose S-200, and anion exchange on
DEAE-cellulose. SAA was ⬎98% pure as determined by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by silver staining. On the day before use, samples were
dissolved in 5M urea and dialyzed overnight in distilled water,
using a 6–8 kd–cutoff dialysis membrane from Spectropor.
Patients. After informed consent was provided, peripheral blood was obtained from patients with RA as classified
according to the American College of Rheumatology (formerly, the American Rheumatism Association) criteria (9).
The levels of endogenous SAA from the patients (n ⫽ 7) were
clearly elevated (mean ⫾ SD 450 ⫾ 175 mg/liter [⬃38 ␮M];
range 200–⬎600 mg/liter [17–⬎50 uM]), as analyzed at the
clinical immunology laboratory of Sahlgrenska University Hos-
RESULTS
Recombinant SAA induces activation of human
neutrophils in whole blood. We monitored neutrophil
activation in whole blood by measuring surface exposure
of the cell adhesion molecule L-selectin. L-selectin is
present on the surface of resting cells, but is rapidly
cleaved off during cell activation. Blood samples from
healthy subjects were stimulated with rSAA or with TNF
(10 ng/ml) as a positive control, and neutrophils were
analyzed by flow cytometry for L-selectin expression
(Figure 1A). Whereas unstimulated cells expressed high
levels of L-selectin, stimulated blood contained neutrophils with cleaved L-selectin, i.e., with an activated
phenotype. Recombinant SAA at the higher concentration used (10 ␮M) induced robust L-selectin shedding
(Figure 1B), which was comparable with the level of
activation induced by the potent neutrophil activator
TNF. Similar results were obtained when neutrophils
were purified and washed before stimulation with rSAA
in buffer (data available at http://hdl.handle.net/2077/
22202). These findings show that rSAA activates human
neutrophils and potently induces L-selectin cleavage in
the presence or absence of blood components, in a
concentration-dependent manner. The biphasic appear-
1662
Figure 1. Effect of recombinant serum amyloid A (rSAA) on neutrophil
activation in whole blood. A, Blood samples from healthy subjects were
stimulated with rSAA (10 ␮M or 0.5 ␮M) or with tumor necrosis factor
(TNF; 10 ng/ml) as a positive control, and flow cytometry was performed
to assess expression of L-selectin. Unstimulated cells expressed L-selectin
in high levels, whereas stimulated blood contained neutrophils with
cleaved L-selectin (region 1). B, L-selectin shedding was assessed by flow
cytometry. Recombinant SAA (10 ␮M) induced robust shedding of
L-selectin, comparable with the level of activation induced by the potent
neutrophil activator TNF (ⴱⴱⴱ ⫽ P ⬍ 0.0001). Cleavage of L-selectin by
rSAA was concentration dependent; at 0.5 ␮M SAA, no significant
cleavage of L-selectin was observed (NS ⫽ not significant). Values are the
mean and SD (n ⫽ 3–7 samples per experimental condition).
ance of the histogram (Figure 1A) is explained by the
fact that very few cells will be intermediary in binding/
fluorescence at any given time.
BJÖRKMAN ET AL
Neutrophils and monocytes from RA patients
with high plasma levels of SAA do not display an
activated phenotype. Next we investigated whether high
levels of SAA in the circulation of RA patients could
activate neutrophils to shed L-selectin. Blood samples
were obtained from the 7 patients with elevated serum
SAA levels and from healthy controls. Without stimulation, neutrophils from both patients and control subjects
were in a resting state (high L-selectin levels) (Figure 2),
indicating that endogenous SAA did not activate neutrophils in the circulation. However, when whole blood
was stimulated with rSAA, complete shedding of
L-selectin from neutrophils was observed (Figure 2).
When monocytes from the same samples were analyzed,
we found that monocytes from RA patients were likewise not activated, but the addition of rSAA to whole
blood led to marked shedding of L-selectin from these
cells as well. Resting neutrophils expressed low levels of
CR3, and this receptor was up-regulated to the cell
surface when the cells were challenged with rSAA or
TNF (data available at http://hdl.handle.net/2077/
22202). Effects were the same in RA patients and
controls, indicating that rSAA activates cells in blood
regardless of how much endogenous SAA is present.
Many proinflammatory effects of SAA have been
reported to be neutralized by the presence of highdensity lipoprotein (HDL) or other blood components
(5). Our data clearly showed that no blood constituents,
either from control or from RA patient blood, were able
to neutralize the effect of rSAA. We also performed
experiments in which rSAA was incubated with human
control plasma (naturally containing HDL) before the
cells were stimulated with the mixture. Such preincubation, for up to 2 hours, did not influence the ability of
rSAA to induce L-selectin cleavage (data available at
http://hdl.handle.net/2077/22202).
Purified SAA obtained from serum during an
acute-phase response does not activate neutrophils or
adherent PBMCs. We then tested whether purified SAA
isolated from patients with an acute-phase response had
the capacity to activate human neutrophils in a similar
manner to that observed with the recombinant protein.
Neither of the purified acute-phase isoforms (SAA1 or
SAA2) caused significant neutrophil activation when
added to blood from healthy subjects (Figure 3A),
whereas rSAA was again very effective at inducing
shedding of L-selectin from these cells. Such effects
were not restricted to selectin expression since other
neutrophil responses seen previously with rSAA, e.g.,
NADPH oxidase activation (7) and the release of
interleukin-8 from purified neutrophils (10), were simi-
SERUM AMYLOID A LACKS PROINFLAMMATORY ACTIVITY
Figure 2. Lack of an activated phenotype in neutrophils from rheumatoid arthritis (RA) patients with high plasma levels of SAA. Blood
samples from 7 RA patients with elevated serum SAA levels (mean ⫾
SD 450 ⫾ 175 mg/liter [⬃38 ␮M]) and from healthy controls were
analyzed for L-selectin expression by flow cytometry. Neutrophils in
unstimulated whole blood from both patients and control subjects were
in a resting state (high L-selectin [red]). Stimulation of whole blood
with rSAA (dark blue) or TNF (10 ng/ml) (light blue), however,
resulted in complete shedding of L-selectin. A, Histograms from a
representative experiment. B, Mean and SD results from 7 independent experiments. See Figure 1 for other definitions.
larly lacking in experiments using purified SAA (data
available at http://hdl.handle.net/2077/22202).
1663
Figure 3. Failure of purified endogenous SAA obtained from serum
during an acute-phase response to activate neutrophils or adherent
peripheral blood mononuclear cells (PBMCs). A, Neutrophils in whole
blood from healthy donors were stimulated with purified endogenous
SAA (SAA1 or SAA2; 8 ␮M). Stimulation with SAA1 or SAA2 did not
result in shedding of L-selectin from the cell surface, whereas rSAA (8
␮M) and TNF (10 ng/ml) potently cleaved L-selectin. Values are the
mean and SD (n ⫽ 3 samples per experimental condition). ⴱⴱⴱ ⫽ P ⬍
0.0001. B, Human adherent PBMCs were stimulated with medium,
lipopolysaccharide (LPS; 1 ␮g/ml) as a positive control, or purified SAA1
or rSAA at various concentrations. After 24 hours, TNF in supernatants
was measured by enzyme-linked immunosorbent assay. Values are the
mean ⫾ SD (n ⫽ 6 samples per experimental condition). ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus control medium. See Figure 1 for
other definitions.
1664
BJÖRKMAN ET AL
We also tested the proinflammatory activity of
the purified SAA preparations under other experimental
conditions in which rSAA has a clear proinflammatory
effect. Whereas rSAA effectively induced TNF production from human monocytes, neither SAA1 (Figure 3B)
nor SAA2 (data not shown) stimulated release of TNF
above background levels.
DISCUSSION
Increased levels of SAA are frequently observed
in the sera of RA patients and have been used as a
sensitive marker for disease activity (11). Several studies
have established a role for SAA in the pathogenesis of
RA as an inflammation mediator, inducing and orchestrating the production of proinflammatory cytokines.
SAA has been proposed to be a key regulator of matrix
degradation, a hallmark of RA, by causing neoangiogenesis and proliferation of synoviocytes (12–15). Various
studies have demonstrated direct receptor-mediated activation of inflammatory leukocytes by rSAA (7,16),
even though the question of receptor usage has been a
matter of debate. The majority of studies investigating
the action of SAA on immune cells have been carried
out using a commercially available recombinant form of
SAA that is a “consensus SAA molecule.”
Human SAA is present in vivo in several different
isoforms, and 2 of these, SAA1 and SAA2, are considered proinflammatory variants, the expression levels of
which increase immensely during the acute-phase response (5). The amino acid sequence of rSAA corresponds to human SAA1 except for the presence of an
N-terminal methionine and substitution of asparagine
for aspartic acid at position 60 and arginine for histidine
at position 71; these substitutions correspond to the
residues found in SAA2, thus forming a hybrid molecule
not identical to any of the human isoforms of SAA. As
mentioned above, this recombinant chimera has been
ascribed numerous different proinflammatory effects
and activates various leukocytes at concentrations well
below those found in the circulation during inflammatory conditions. We thus reasoned that leukocytes obtained from SAA-rich blood would be activated, and we
used peripheral blood from RA patients to investigate
this.
Cleavage of L-selectin from the cell surface is an
early activation marker for neutrophils that proceeds in
vivo by the time the phagocytes adhere to the vessel wall
and initiate transmigration to the tissues (17). Our data
show that in vitro addition of rSAA to blood at concentrations between 0.5 ␮M and 10 ␮M potently activated
neutrophils to shed L-selectin. However, neutrophils
constantly subjected to up to 50 ␮M of endogenous SAA
in the circulation of RA patients were not activated in
terms of L-selectin cleavage. An undesired effect of
using recombinant (i.e., bacterial-derived) proteins is
that proinflammatory effects could be caused by contamination with endotoxin, rather than the recombinant
compound itself. For example, the proinflammatory
effect of recombinant human C-reactive protein has
been shown to be caused by contamination with bacterial products (e.g., endotoxin) and not by C-reactive
protein itself (18). The rSAA preparation used in this
study contained very low endotoxin levels. Also, rSAA
activates neutrophils through a G protein–coupled receptor (7,16) and induces cellular activities (e.g., activation of NADPH oxidase and mobilization of intracellular calcium) not triggered by endotoxin. Nevertheless,
we cannot at present entirely rule out the possibility that
biologically active bacterial molecules other than endotoxin could accompany the rSAA.
Physiologically, it makes sense that neutrophils
already in the circulation would not be activated. It is,
however, clear that under certain conditions (e.g., sepsis), L-selectin can be cleaved off of neutrophils already
in the circulation (Karlsson A, et al: unpublished observations). In addition, stimulation of whole blood (with
TNF or rSAA) very clearly activated the cells in this
environment, indicating that the shedding machinery is
intact and functional. A potential mechanism for avoidance of premature cellular activation by endogenous
SAA in the circulation could be that some additional
blood component, capable of neutralizing the proinflammatory actions of SAA, is also present in the circulation.
Such neutralizing effects have been described for HDL,
and SAA is present in plasma mainly as an HDLassociated apolipoprotein (5). However, spiking the
already SAA-rich blood with rSAA induced potent
neutrophil activation, indicating that HDL or other
factors present in the blood failed to neutralize the
biologic activity of rSAA. To exclude the possibility that
the high levels of endogenous SAA could have saturated
the HDL in patient plasma, we incubated rSAA with
control plasma (naturally containing HDL) for up to 2
hours, in order to facilitate potential binding to neutralizing blood constituents, without dampening the
neutrophil-activating potential. The use of multiple different RA donors and different donor sources of purified SAA rules out differences that may have been due
to allelic variants of SAA.
Taken together, our findings demonstrate that
rSAA differs functionally from SAA found in the circu-
SERUM AMYLOID A LACKS PROINFLAMMATORY ACTIVITY
lation during inflammatory conditions, and the latter
lack neutrophil-activating capacity. The purification process (both for the recombinant as well as for the
acute-phase SAA) might render the protein to a nonphysiologic state, although previous work with endogenous SAA, purified or as present in the circulation, has
demonstrated its ability to mediate phagocyte recognition of gram-negative bacteria, i.e., SAA functions as an
opsonin (19). Even though our data show that the SAA
present in the circulation during an acute-phase response lacks direct proinflammatory effects (such as
activation of neutrophils), it could still be of functional
importance as an opsonin during gram-negative sepsis.
It should be noted that SAA is a protein that is
prone to aggregation and can assemble in extracellular
amyloid plaques in the tissues and have deleterious
effects on organ functions (4). The removal of SAA from
its carrier HDL may give rise to a form (frequently
termed “lipid poor”) that is predisposed to change
conformation, potentially in multiple different ways; for
instance, a hexameric form has been described (20). For
the comparison of SAA preparations used in this study,
it was essential that they showed similar aggregation
status by gel filtration analysis. The preparations of
SAA1, SAA2, and rSAA did exhibit similar aggregation
status, providing evidence that aggregation alone is not
sufficient to cause activation of neutrophils.
The details of SAA aggregation are still largely
unelucidated, as is the aggregation state of endogenous
SAA found at different sites. Hopefully, future studies
will explore whether proinflammatory forms of endogenous SAA exist outside of the circulation, for example,
in inflamed RA joints.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Björkman had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Björkman, Raynes, Shah, Karlsson,
Dahlgren, Bylund.
Acquisition of data. Björkman, Shah, Bylund.
Analysis and interpretation of data. Björkman, Raynes, Karlsson,
Bylund.
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